ORIGINAL RESEARCH
Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression Dongdong Wang, PhD;* Anela Tosevska, PhD;* Elke H. Heiß, PhD; Angela Ladurner, PhD; Christine M€ olzer, PhD; Marlies Wallner, PhD; Andrew Bulmer, PhD; Karl-Heinz Wagner, PhD; Verena M. Dirsch, PhD;† Atanas G. Atanasov, PhD†
Background-—Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. Methods and Results-—Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3–17.1 lmol/L) exogenously added to plasma- or apolipoprotein A1–supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1–mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. Conclusions-—Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin’s impact on cholesterol transport and represent an important advancement in our understanding of bilirubin’s role in cardiovascular disease. ( J Am Heart Assoc. 2017;6:e005520. DOI: 10.1161/JAHA.117.005520.) Key Words: ATP-binding cassette transporter • bilirubin • cardiovascular disease • cholesterol • cholesterol homeostasis
F
or many years, bilirubin, the end product of heme catabolism, was regarded as a harmful waste product1
From the Departments of Pharmacognosy (D.W., E.H.H., A.L., V.M.D., A.G.A.) and Nutritional Sciences (A.T., C.M., M.W., K.-H.W.), and Research Platform Active Ageing (A.T., K.-H.W.), University of Vienna, Austria; Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, Jastrzebiec, Poland (D.W., A.G.A.); Department of Molecular, Cell and Developmental Biology, UCLA, Los Angeles, CA (A.T.); School of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of Aberdeen, United Kingdom (C.M.); Institute of Dietetics and Nutrition, University of Applied Sciences, FH JOANNEUM, Graz, Austria (M.W.); School of Medical Science and Menzies Health Institute Queensland, Gold Coast, Australia (A.B.). *Dr Wang and Dr Tosevska contributed equally to this work and are co-first authors. † Dr Dirsch and Dr Atanasov contributed equally to this work and are co-last authors Correspondence to: Atanas G. Atanasov, PhD, Department of Pharmacognosy, University of Vienna, Althanstrasse 14, Vienna A-1090, Austria. E-mail: [email protected] Received January 19, 2017; accepted March 17, 2017. ª 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
DOI: 10.1161/JAHA.117.005520
and was assumed to possess little or no biological function.2 More recent evidence, however, suggests an inverse relationship between circulating unconjugated bilirubin (UCB) and the risk of cardiovascular disease (CVD).1 In 1994, Schwertner et al3 first reported that serum bilirubin concentrations in the upper physiological range (10–17.1 lmol/L)3 were associated with reduced prevalence of coronary artery disease. Subsequently, a number of retrospective and prospective studies indicated that high-normal (10–17.1 lmol/L)3 serum bilirubin is associated with decreased risk of CVD.4–11 These reports, however, do not conclusively implicate a cardiovascular protective effect of mildly elevated UCB concentrations and demonstrate associations between cardiovascular risk factors in cohorts of subjects with Gilbert syndrome (GS), in the range of 17.1 to 90 lmol/L.12 GS is a genetic disorder resulting in a benign, mildly elevated UCB serum concentration with a prevalence of 5% to 10% in the white population.2,12,13 The main cause of GS is a hereditary mutation in the promoter of the uridine diphosphateglucuronosyltransferase (UGT) gene, which leads to reduced hepatic UGT 1 family, polypeptide A1 (UGT1A1) expression.14 Journal of the American Heart Association
1
Bilirubin Decreases Cholesterol Efflux
Wang et al
DOI: 10.1161/JAHA.117.005520
important steps in the atherosclerotic process. In addition, patients with GS are reported to have improved resistance to serum oxidation,32,34 altered inflammatory responses,35,36 and modified lipid status and metabolism,2,41–43 all of which likely contribute to cardiovascular protection in GS. Similar protective effects were also demonstrated in the Gunn rats.2,10,44,45 Although bilirubin appears to affect multiple steps in the atherosclerotic process, it remains to be established whether variations of UCB plasma concentrations influence macrophage cholesterol efflux, which is a promising target for the prevention and treatment of CVD.41,42 Clinical reports indicate that macrophage cholesterol efflux is significantly and inversely associated with CVD, independent of high-density lipoprotein cholesterol (HDL-C) concentrations,41–43 suggesting that the cholesterol efflux capacity may be a novel predictive biomarker for the incidence of cardiovascular events.46 A well-established pathway of macrophage cholesterol efflux involves apolipoprotein A1 (apo A1; the major protein in HDL) as an acceptor and membrane-associated transporter ATP-binding cassette transporter A1 (ABCA1).47,48 ABCA1 promotes cholesterol efflux from macrophages to lipid-poor apo A1 (often referred to as pre-b HDL).49 ABCA1 is confirmed as a major mediator of cholesterol efflux to HDL.50,51 In the current study, we compared cholesterol efflux mediated by plasma from patients with GS or Gunn rats with that from normobilirubinemic humans and rats, respectively. Moreover, we evaluated the influence of UCB on macrophage cholesterol efflux and ABCA1 protein expression and degradation.
Materials and Methods Human Participants Sixty patients with GS and 60 age- and sex-matched healthy controls were recruited according the inclusion and exclusion criteria described by M€olzer et al52 and Tosevska et al.53 Methods used for plasma sample preparation, peripheral blood mononuclear cell (PBMC) isolation, age distribution, UCB levels, and blood biochemistry were described by M€ olzer et al.52 The study was approved by the ethics committee of the Medical University of Vienna and the General Hospital of Vienna (no. 1164/2014) and conducted in accordance with the guidelines approved by the Declaration of Helsinki. All participants provided written informed consent.
Animals Plasma samples from 20 hyperbilirubinemic Gunn rats (homozygous for a mutation in UGT1A1, 10 male and 10 female) and 20 respective controls (10 male and 10 female), normobilirubinemic Wistar rats (heterozygous for a mutation Journal of the American Heart Association
2
ORIGINAL RESEARCH
UGT1A1 is primarily responsible for conjugating bilirubin with glucuronic acid, allowing its excretion via the bile from the circulation.15 In addition to bilirubin, UGT1A1 is involved in the glucuronidation of 17b-estradiol, 17a-ethinylestradiol, and some xenobiotics.16 Nevertheless, no evidence currently exists to demonstrate that this UGT1A1 polymorphism affects hormone levels in postmenopausal women.17 Interestingly, patients with GS have significantly reduced incidence of ischemic heart disease18 and significant delay in developing clinically relevant manifestations of CVD19 compared with the general population. Furthermore, homozygous carriers of the UGT1A1*28 allele, characterized by the presence of an additional TA repeat in the TATA sequence of the UGT1A1 promoter—(TA)7TAA instead of (TA)6TAA)8— with mildly elevated serum UCB concentrations, also demonstrate a reduced risk of CVD.8 Elevated serum UCB concentrations are also reported in Gunn rats.18 Gunn rats inherit a single point mutation in the coding region of the UGT1A1 gene that truncates and inactivates UGT1A1, leading to complete absence of bilirubin glucuronidation capacity.2,20 UCB serum concentrations of these animals range between 50 and 200 lmol/L.21 In line with observations in human GS, hyperbilirubinemia beneficially modulated myocardial function and aortic ejection and imparted ischemic stress resistance in Gunn rats.22 Although a body of evidence indicates that upper normal (10–17.1 lmol/L)3 or mildly elevated (17.1–90 lmol/L)12 plasma bilirubin levels are associated with a reduced risk of CVD, conflicting reports show varying binomial relationships,23–28 with a recent report suggesting that humans with higher bilirubin levels (12–86 lmol/L)29 have a risk similar to that of persons with the lowest bilirubin levels (
Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression Dongdong Wang, PhD;* Anela Tosevska, PhD;* Elke H. Heiß, PhD; Angela Ladurner, PhD; Christine M€ olzer, PhD; Marlies Wallner, PhD; Andrew Bulmer, PhD; Karl-Heinz Wagner, PhD; Verena M. Dirsch, PhD;† Atanas G. Atanasov, PhD†
Background-—Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. Methods and Results-—Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3–17.1 lmol/L) exogenously added to plasma- or apolipoprotein A1–supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1–mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. Conclusions-—Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin’s impact on cholesterol transport and represent an important advancement in our understanding of bilirubin’s role in cardiovascular disease. ( J Am Heart Assoc. 2017;6:e005520. DOI: 10.1161/JAHA.117.005520.) Key Words: ATP-binding cassette transporter • bilirubin • cardiovascular disease • cholesterol • cholesterol homeostasis
F
or many years, bilirubin, the end product of heme catabolism, was regarded as a harmful waste product1
From the Departments of Pharmacognosy (D.W., E.H.H., A.L., V.M.D., A.G.A.) and Nutritional Sciences (A.T., C.M., M.W., K.-H.W.), and Research Platform Active Ageing (A.T., K.-H.W.), University of Vienna, Austria; Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, Jastrzebiec, Poland (D.W., A.G.A.); Department of Molecular, Cell and Developmental Biology, UCLA, Los Angeles, CA (A.T.); School of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of Aberdeen, United Kingdom (C.M.); Institute of Dietetics and Nutrition, University of Applied Sciences, FH JOANNEUM, Graz, Austria (M.W.); School of Medical Science and Menzies Health Institute Queensland, Gold Coast, Australia (A.B.). *Dr Wang and Dr Tosevska contributed equally to this work and are co-first authors. † Dr Dirsch and Dr Atanasov contributed equally to this work and are co-last authors Correspondence to: Atanas G. Atanasov, PhD, Department of Pharmacognosy, University of Vienna, Althanstrasse 14, Vienna A-1090, Austria. E-mail: [email protected] Received January 19, 2017; accepted March 17, 2017. ª 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
DOI: 10.1161/JAHA.117.005520
and was assumed to possess little or no biological function.2 More recent evidence, however, suggests an inverse relationship between circulating unconjugated bilirubin (UCB) and the risk of cardiovascular disease (CVD).1 In 1994, Schwertner et al3 first reported that serum bilirubin concentrations in the upper physiological range (10–17.1 lmol/L)3 were associated with reduced prevalence of coronary artery disease. Subsequently, a number of retrospective and prospective studies indicated that high-normal (10–17.1 lmol/L)3 serum bilirubin is associated with decreased risk of CVD.4–11 These reports, however, do not conclusively implicate a cardiovascular protective effect of mildly elevated UCB concentrations and demonstrate associations between cardiovascular risk factors in cohorts of subjects with Gilbert syndrome (GS), in the range of 17.1 to 90 lmol/L.12 GS is a genetic disorder resulting in a benign, mildly elevated UCB serum concentration with a prevalence of 5% to 10% in the white population.2,12,13 The main cause of GS is a hereditary mutation in the promoter of the uridine diphosphateglucuronosyltransferase (UGT) gene, which leads to reduced hepatic UGT 1 family, polypeptide A1 (UGT1A1) expression.14 Journal of the American Heart Association
1
Bilirubin Decreases Cholesterol Efflux
Wang et al
DOI: 10.1161/JAHA.117.005520
important steps in the atherosclerotic process. In addition, patients with GS are reported to have improved resistance to serum oxidation,32,34 altered inflammatory responses,35,36 and modified lipid status and metabolism,2,41–43 all of which likely contribute to cardiovascular protection in GS. Similar protective effects were also demonstrated in the Gunn rats.2,10,44,45 Although bilirubin appears to affect multiple steps in the atherosclerotic process, it remains to be established whether variations of UCB plasma concentrations influence macrophage cholesterol efflux, which is a promising target for the prevention and treatment of CVD.41,42 Clinical reports indicate that macrophage cholesterol efflux is significantly and inversely associated with CVD, independent of high-density lipoprotein cholesterol (HDL-C) concentrations,41–43 suggesting that the cholesterol efflux capacity may be a novel predictive biomarker for the incidence of cardiovascular events.46 A well-established pathway of macrophage cholesterol efflux involves apolipoprotein A1 (apo A1; the major protein in HDL) as an acceptor and membrane-associated transporter ATP-binding cassette transporter A1 (ABCA1).47,48 ABCA1 promotes cholesterol efflux from macrophages to lipid-poor apo A1 (often referred to as pre-b HDL).49 ABCA1 is confirmed as a major mediator of cholesterol efflux to HDL.50,51 In the current study, we compared cholesterol efflux mediated by plasma from patients with GS or Gunn rats with that from normobilirubinemic humans and rats, respectively. Moreover, we evaluated the influence of UCB on macrophage cholesterol efflux and ABCA1 protein expression and degradation.
Materials and Methods Human Participants Sixty patients with GS and 60 age- and sex-matched healthy controls were recruited according the inclusion and exclusion criteria described by M€olzer et al52 and Tosevska et al.53 Methods used for plasma sample preparation, peripheral blood mononuclear cell (PBMC) isolation, age distribution, UCB levels, and blood biochemistry were described by M€ olzer et al.52 The study was approved by the ethics committee of the Medical University of Vienna and the General Hospital of Vienna (no. 1164/2014) and conducted in accordance with the guidelines approved by the Declaration of Helsinki. All participants provided written informed consent.
Animals Plasma samples from 20 hyperbilirubinemic Gunn rats (homozygous for a mutation in UGT1A1, 10 male and 10 female) and 20 respective controls (10 male and 10 female), normobilirubinemic Wistar rats (heterozygous for a mutation Journal of the American Heart Association
2
ORIGINAL RESEARCH
UGT1A1 is primarily responsible for conjugating bilirubin with glucuronic acid, allowing its excretion via the bile from the circulation.15 In addition to bilirubin, UGT1A1 is involved in the glucuronidation of 17b-estradiol, 17a-ethinylestradiol, and some xenobiotics.16 Nevertheless, no evidence currently exists to demonstrate that this UGT1A1 polymorphism affects hormone levels in postmenopausal women.17 Interestingly, patients with GS have significantly reduced incidence of ischemic heart disease18 and significant delay in developing clinically relevant manifestations of CVD19 compared with the general population. Furthermore, homozygous carriers of the UGT1A1*28 allele, characterized by the presence of an additional TA repeat in the TATA sequence of the UGT1A1 promoter—(TA)7TAA instead of (TA)6TAA)8— with mildly elevated serum UCB concentrations, also demonstrate a reduced risk of CVD.8 Elevated serum UCB concentrations are also reported in Gunn rats.18 Gunn rats inherit a single point mutation in the coding region of the UGT1A1 gene that truncates and inactivates UGT1A1, leading to complete absence of bilirubin glucuronidation capacity.2,20 UCB serum concentrations of these animals range between 50 and 200 lmol/L.21 In line with observations in human GS, hyperbilirubinemia beneficially modulated myocardial function and aortic ejection and imparted ischemic stress resistance in Gunn rats.22 Although a body of evidence indicates that upper normal (10–17.1 lmol/L)3 or mildly elevated (17.1–90 lmol/L)12 plasma bilirubin levels are associated with a reduced risk of CVD, conflicting reports show varying binomial relationships,23–28 with a recent report suggesting that humans with higher bilirubin levels (12–86 lmol/L)29 have a risk similar to that of persons with the lowest bilirubin levels (
